Human cytomegalovirus (HCMV) is a member of the Herpesviruses. This widespread pathogen is responsible for causing asymptomatic and persistent infections. Additional herpesviruses include, herpes simplex-1 and -2, varicella-zoster, Epstein-Barr, and Kaposi's sarcoma-associated herpesvirus. HCMV has a large impact on medical and public health by being the leading cause of congenital infection in the United States (Fowler, McCollister et. al. 1998), with an estimated 1% of newborns being infected annually with HCMV. This is approximately 40,000 new cases each year (Fowler, McCollister et. al. 1998). Of these cases, 90% to 95% will hot display symptoms, but can still have central nervous system and sensory impairments. Antivirals approved for HCMV are limited and target the same step in HCMV infection, DNA synthesis. The halogenated benzimidazole maturational inhibitors TCRB (2,5,6-trichloro-1- (3-D- ribofuranosyl) benzimidazole) and BDCRB (2-bromo-5, 6-dichloro-1- (p-D-ribofuranosyl) benzimidazole) block cleavage of HCMV concatemeric DNA into 230-kb genomes while also resulting in formation of a novel 270-kb species termed "monomer-plus" (M+) (Underwood, Harvey et. al. 1998 and Krosky, Underwood et. al. 1998). Preliminary studies indicate that this novel DNA species does not contain a terminal left end and is nuclease-sensitive. These results can be explained by the skipping of the proper cleavage site and continuation of packaging the DNA until the next cleavage site. This would create a short- long-short structure of 270-kb in length. To test this hypothesis, the specific aims are designed to (1) examine the structure of M+ DNA by restriction analysis, (2) determine M+ dependence on the presence of internal cleavage sites by deleting the internal junction of the virus, and (3) determine if M+ DNA is encapsidated by capsid purification and electron microscopy. Understanding the mechanism by which BDCRB inhibits packaging and cleavage to cause defective genomes is beneficial in understanding how genomes enter capsids, understanding the function of a terminase complex, and in developing new antivirals that can target packaging and cleavage of viral DNA. This project is relevant to public health because it will enhance the basic understanding of the mechanism of action of the drug BDCRB and enhance the current knowledge of how viral genomes are packaged into viral capsids. Insight into viral packaging mechanisms will benefit public health by furthering development of antivirals for HCMV and perhaps other herpes viruses. The current available antivirals target viral DNA synthesis, which can cause cross-resistance when treating patients.